The mechanisms of drug and hormone action on glycogenolysis are being investigated using a single gene, sex-linked variant (carried in I strain mice) that results in a marked deficiency in phosphorylase b kinase (PBK) activity. As assayed in muscle extracts I strain had 0.3% of the PBK activity found in the control strains C57BI/St and Ha/ICR (a Swiss-Webster strain). Nevertheless I strain mouse skeletal muscle contained protein that cross reacted with an antiserum to rabbit skeletal muscle PBK, indicating the presence of a structurally altered enzyme protein. The mutant activity was shown to have the same pH optimum, Km for phosphorylase b, and Mg2 ion-ATP concentration dependence as the control strain PBK activity. The mutant phosphorylase b to a converting activity incorporated P32 from (gamma-P32)ATP into phosphorylase and the labelled phosphorylase a was isolated. The addition of ATP (10 mM), MG2 ion (50 mM), and Ca2 ion (4mM) to a protein-glycogen complex isolated by differential centrifugation of I strain skeletal muscle extracts increased phosphorylase activity as the -AMP: plus AMP ratio from 0.021 to 0.075 within 20 secs as compared to an increase in activity from 0.08 to 0.85 in the protein-glycogen complex isolated from the control strain. In addition incubation of diaphragm muscle from PBK deficient animals with 4 micron M isoproterenol for 10 minutes produced a conversion of phosphorylase b to a. The -AMP: plus AMP ratio increased from .043 to .078 in mutant diaphragms as compared to .060 to .168 in the control strain. Thus functional PBK activity exists in skeletal muscle of animals with the PBK deficiency mutation. Since epinephrine stimulates glycogen breakdown in I strain gastrocnemius muscle, conversion of phosphorylase b to a may be a necessary step in catecholamine-stimulated glycogenolysis in I strain skeletal muscle as it is in normal skeletal muscle.